The present invention relates to a ligand for the cell-surface antigen CD40, CD40 ligand (CD40L). More specifically, the present invention relates to methods of detecting mutations in a CD40L gene, and to methods of treating a syndrome that results in elevated levels of serum IgM and diminished levels of all other isotypes of immunoglobulins.
Human X-linked hyper-IgM syndrome is characterized by an elevated level of serum IgM and diminished (virtually undetectable) levels of other isotypes of immunoglobulins. Affected males usually experience onset of recurrent infection in the first year of life. The clinical course may include intermittent neutropenia and Pneumocystis pneumonia, as well as infections that are more typical of hypogammaglobulinemia, such as bacterial otitis, sinusitis, and pneumonia. This condition is lethal in the absence of medical intervention; however, patients typically respond well to a maintenance therapy consisting of intravenous gamma globulin, especially if therapy is initiated soon after birth.
Affected males have normal numbers of circulating B and T lymphocytes, although lymph node hyperplasia with an absence of germinal centers is common (Notarangelo et al., Annu. Rev. Immunol. 10:215, 1992). B-cells from such patients appear to be normal in that they can be induced to undergo isotype switching when cultured in vitro with a T cell line known to induce class switching in normal B-cell cultures (Hendricks et al., Eiur. J. Immunol. 20:2603, 1990; Mayer et al., N. Engl. J. Med. 314:409, 1986).
Elevated levels of serum IgM occur in other syndromes, including combined variable immune deficiency (CVID) and post congenital rubella. Alterations of T-cell activation either as a result of primary genetic immune deficiency or acquired CD4+ T-cell abnormality (e.g. AIDS) may also cause loss of CD40L-induced B-cell activation signals and thus partially explain the secondary abnormalities of B-cell function observed in these conditions.
The CD40 cell surface antigen has been shown to play an important role in B-cell proliferation and differentiation. Human CD40 protein (CD40), a cell-surface antigen present on the surface of B cells, is a peptide of 277 amino acids having a molecular weight of 30,600, with a 19 amino acid secretory signal peptide comprising predominantly hydrophobic amino acids. A cDNA encoding human CD40 was isolated from a cDNA library prepared from Burkitt lymphoma cell line Raji (Stamenkovic et al., EMBO J. 8:1403, 1989).
Activated CD4+ T cells express high levels of a ligand for CD40 (CD40L). Human CD40L, a membrane-bound glycoprotein, has recently been cloned from peripheral blood T-cells as described in Spriggs et al., J.Exp. Med. 176:1543 (1992), and in U.S. patent application Ser. No. 07/969,703, filed Oct. 23, 1992, the disclosure of which is incorporated by reference herein. The cloning of murine CD40L is described in Armitage et al., Nature 357:80, 1992. CD40L induces B-cell proliferation and secretion of various immunoglobulin isotypes (except IgE) in the absence of any co-stimulus, and can also induce production of IgE in the presence of cytokines.
CD40L thus appears to play a critical role in the cognate interaction between CD4+ T helper cells and B cells. A more detailed analysis of patients with X-linked hyper IgM syndrome and its related syndromes will provide valuable information on the T cell-B cell interactions involved in the humoral immune response. Early detection of X-linked hyper IgM syndrome and its related syndromes will allow prompt initiation of appropriate therapy.
Therefore, there is a need in the art to develop methods of detecting and conflrming X-linked hyper IgM syndrome and other abnormalities in B cell-T cell interactions in which CD40 and CD40L play a role. Alternative methods of treatment of such syndromes are also needed.
The present invention relates to methods of detecting a mutation or mutations in a CD40L gene, comprising isolating nucleic acid (RNA or DNA) from an individual, selectively amplifying nucleic acid derived from the CD40 ligand gene and analyzing the amplified nucleic acid to determine if there is a mutation (or mutations) in the CD40L gene. Mutations in this gene result in abnormalities in the interaction of CD40L and CD40, which result in alterations in the interactions of T cells and B cells. Such alterations in T cell-B cell interaction play a role in X-linked hyper-IgM syndrome in a human, and may also occur in other syndromes.
The present invention further provides a method of treating an individual that has a syndrome in which the interaction of T cells and B cells is affected (such as X-linked hyper-IgM syndrome), comprising administering an effective amount of a soluble CD40L. Soluble forms of CD40L comprise the extracellular region of CD40L, and include, for example, fusion proteins comprising the extracellular region of CD40L and an Fc region of a human immunoglobulin, and CD40L multimers formed by adding a multimer-forming peptide to the extracellular region of CD40L.
The present invention also provides a method of utilizing gene therapy to correct X-linked hyper-IgM syndrome and other syndromes in which the CD40L gene does not encode biologically active CD40L. Gene therapy to correct such syndromes comprises isolating CD4+ T cells from an affected individual, transfecting the isolated T cells with a transfection vector that expresses a biologically active CD40L, and administering the transfected T cells expressing biologically active CD40L to the individual.
Also provided are animals that, through gene targeting technology utilizing embryonic stem cells, express non-functional CD40-L in vivo. Such animals, which are referred to as knockout animals, provide an non-human model useful is studying the cognate interaction of T and B cells in vivo.